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Vol. 14, Issue 11, 4654-4666, November 2003
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Unit of Molecular and Cellular Biology, Royal Veterinary College, University of London, London NW1 0TU, United Kingdom
Submitted March 31, 2003;
Revised July 9, 2003;
Accepted July 15, 2003
Monitoring Editor: Paul Matsudaira
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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; Gordon-Weeks, 2000). Growth cone advance occurs through adhesive interactions with the surrounding extracellular matrix in response to external guidance cues; such movement is the result of two alternating processes, outgrowth and retraction, necessary to achieve net protrusive advance (Kater and Guthrie, 1990; Letourneau et al., 1991; Gordon-Weeks, 2000). Elaboration and subsequent disassembly of focal contacts at the leading edge of the growth cone in response to Rac- and Rho-dependent signaling pathways (Hall, 1998; Luo, 2000) brings about changes in trajectory. Such regulatory cascades target the activity of molecular motors and the arrangement of the actin cytoskeleton, which determine growth cone directionality.
The neuronal growth cone comprises two very distinct regions (Bridgman and Dailey, 1989; Lewis and Bridgman, 1992): a relatively massive central domain (C), containing the bulk of the actin cytoskeleton in which actin is arranged as a three-dimensional meshwork, and a thin peripheral region (P), encompassing the lamellipodium and filopodia. Filamentous actin within the P domain originates at the leading edge and is propelled backwards by retrograde actin flow (Lin and Forscher, 1995). Lamellipodial protrusion, as distinct from neuritic process outgrowth, is a dynamic activity localized entirely within the P domain; it is driven by dendritic nucleation under the control of Arp 2/3 and does not necessarily require myosin action (Pantalini et al., 1999; Borisy and Svitkina, 2000), although myosin participation is not excluded (Wang et al., 1996; Diefenbach et al., 2002). However, much greater forces are required to achieve neuritic process dynamics and these forces operate within the C domain and at its periphery with the P domain to move the bulk of the growth cone cytoskeleton. Actin microfilaments constitute the bulk of the cytoskeleton in this region (Chang and Goldman, 1973; Letourneau, 1981; Lewis and Bridgman, 1992), and consequently, actin-based motors are likely to be responsible for fundamental aspects of growth cone movement.
The myosin superfamily comprises at least 18 separate classes of molecule (Berg et al., 2001). Members of the "conventional" class II myosins (Kuczmarski and Rosenbaum, 1978; Cheng et al., 1992; Miller et al., 1992; Li et al., 1994; Rochlin et al., 1995; Ruchhoeft and Harris, 1997) as well as isoforms of "uncoventional" myosins of classes I (Li and Chantler, 1992; Miller et al., 1992; Wagner et al., 1992; Lewis and Bridgman, 1996; Wang et al., 1996), V (Espreafico et al., 1992; Wang et al., 1996; Coling et al., 1997), VI (Suter et al., 2000), and X (Berg and Cheney, 2002) have been found in neuronal growth cones. It would seem likely that these different isoforms perform distinct tasks during the normal functioning of the cell. However, although myosin V (Wang et al., 1996) and myosin 1c (Diefenbach et al., 2002) have been implicated in lamellipodial dynamics, and myosin X in intrafilopodial motility, it would seem that the forces responsible for neurite extension are generated by conventional myosin isoforms (Wylie et al., 1998; Bridgman et al., 2001; Wylie and Chantler, 2001).
At least two different isoforms of conventional myosin, expressed from separate genes (A and B), are found in neuronal cells (Katsuragawa et al., 1989; Kawamoto and Adelstein, 1991; Simons et al., 1991), myosin IIB being the predominate isoform (Katsuragawa et al., 1989; Kawamoto and Adelstein, 1991; Miller et al., 1992; Itoh and Adelstein, 1995; Rochlin et al., 1995). These isoforms exhibit differential localization within the growth cone; myosin IIB, found in the P-domain and adjacent to the C-domain, exhibits a more peripheral distribution than myosin IIA, which is located mainly within the C-domain and does not extend into the marginal zone (Rochlin et al., 1995). Isoform sorting of these conventional myosins may occur by mechanisms intrinsic to their structures (Kolega, 1998). Using antisense knock-down and transgenic knock-out approaches, respectively, we (Wylie et al., 1998) and others (Bridgman et al., 2001) have identified myosin IIB as the molecular motor responsible for neurite outgrowth. We have also shown that myosin IIA, although not directly involved in outgrowth per se, is required for assembly of focal contacts that provide adhesion (Wylie and Chantler, 2001), against which the forward propulsive force acts (Lamoureux et al., 1989). If myosin IIB drives outgrowth and myosin IIA is responsible for tension maintenance during adhesion, it remains to determine what kind of motor is responsible for driving neurite retraction. Here, using an isoform-specific functional knock-down approach combined with pharmacological targeting, we show that myosin IIA, but not myosin IIB, drives neurite retraction.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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; Wylie and Chantler, 2001). Cytochemistry was preceded by a brief extraction step (Cramer and Mitchison, 1995) in cytoskeletal buffer (10 mM MES pH 6.1, 138 mM KCl, 3 mM MgCl2, 2 mM EGTA) supplemented with 0.32 M sucrose, 0.1% Triton X-100, and 1 µg/ml phalloidin, followed by fixation for 30 min in cytoskeletal buffer supplemented with 0.32 M sucrose and 4% formaldehyde, as described previously (Wylie and Chantler, 2001). Cells were permeabilized for 10 min in PBS plus 0.5% Triton X-100, rinsed in phosphate-buffered saline (PBS), and then immersed in a blocking solution comprising 2% decomplemented horse serum in PBS for 20 min. Incubation with primary and secondary antibodies was performed as described previously (Wylie and Chantler, 2001). Nonmuscle myosin IIA was detected by indirect immunofluorescence using polyclonal rabbit anti-nonmuscle (rat) myosin IIA as primary antibody (Choi et al., 1996) (1:300 dilution) and Alexa-Fluor 633-conjugated goat antirabbit IgG (Molecular Probes, Eugene, OR) (1:100 dilution) as secondary antibody. Paxillin was detected by direct immunofluorescence using a fluorescein isothiocyanate-conjugated monoclonal mouse anti-paxillin IgG (BD Biosciences, San Jose, CA) (1:100 dilution)(Leventhal and Feldman, 1996). Filamentous actin was detected using Texas Red phalloidin (Molecular Probes) added at a concentration of 165 nM. All antibody dilutions were made up in 1% decomplemented horse serum/PBS including 0.1% Triton X-100/1.5 mM sodium azide. Coverslips were mounted using MOWIOL (Calbiochem, San Diego, CA)/glycerol [25% (vol/vol)]/50 mM Tris pH 8.5/1,4-diazabicyclo[2-2-2]octane (DABCO) (Sigma) [2.5% (wt/vol)]/3 mM sodium azide.
Antisense Treatment Protocols
Sequences of the isoform-specific antisense and sense oligonucleotides used in these studies, together with the scrambled (control) oligonucleotides used, are displayed in Table 1. For myosin IIA, sense oligonucleotides (AQ5) were synthesized to correspond to a region of isoform-specific sequence located toward the 5' end of the transcript, where it normally codes for the amino acid sequence NPILEA (accession no. U31463
[GenBank]
; Choi et al., 1996). The antisense oligonucleotide sequence (AQ3) was the inverse complement of AQ5. For myosin IIB, sense oligonucleotides (BQ5) were synthesized to correspond to a region of isoform-specific sequence within the 5' coding region of the myosin IIB transcript that codes for the amino acid sequence ADPILES (accession no. U15766
[GenBank]
; Itoh and Adelstein, 1995). The antisense oligonucleotide sequence (BQ3) was the inverse complement of BQ5. Scrambled (control) oligonucleotides corresponded in base composition to those of the antisense oligonucleotides except that the sequence was scrambled and did not match any other entry in the GenBank or European Molecular Biology Laboratory databases.
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Neuro-2A cells in culture were transferred to serum-free media and treated separately, or in combination, with sense, antisense, or scrambled oligonucleotides derived from myosin IIA or myosin IIB sequences. Initial incubations took place with 50 µM oligonucleotides, whereas subsequent additions, every 12 h, were at 25 µM (Wylie et al., 1998). At appropriate times, cells were fixed then examined by differential interference contrast (DIC) microscopy or stained with antibodies before viewing by confocal laser scanning microscopy. Where performed, reverse transcription-polymerase chain reaction was carried out as described previously (Wylie et al., 1998; Wylie and Chantler, 2001).
Confocal Laser Scanning and DIC Microscopy
Confocal fluorescence microscopy was performed, as described previously (Wylie and Chantler, 2001), by using an LSM 510 confocal laser scanning microscope (Carl Zeiss, Jena, Germany) equipped with a 40x Fluar oil immersion objective (numerical aperture 1.3), argon (
ex = 488 nm), and He-Ne (ex = 543 nm) lasers. Neurite outgrowth was monitored through DIC microscopy using an Axiovert 135 inverted microscope (Carl Zeiss). Images were digitized before neurite length measurement using Kontron 300 software. Statistical analysis was performed as described previously (Wylie et al., 1998; Wylie and Chantler, 2001).
Application of Lysophosphatidate (LPA), Thrombin, and Y27632: Serial Measures on Individual Neurites
LPA (1 µM; Sigma-Aldrich, St. Louis, MO) and thrombin (5 NIH units [75 µg/ml]; Sigma-Aldrich) were freshly prepared in media from appropriately diluted stock solutions and added as a 20-µl pulse to the cultured cells, in a preselected quadrant of the coverslip under observation (Jalink and Moolenaar, 1992; Suidan et al., 1992), initiating retraction. In those experiments where the Rho-kinase inhibitor Y27632 (50 µM; Calbiochem) was required, it was added 30 min before the addition of either LPA or thrombin.
For serial measures, the same patch of cells was monitored using DIC optics from the start of the timed series, digitized images (Kontron KS-300) being stored at the following intervals: 0, 2, 5, 10, 15, 30, 60, and 90 min. The microscope stage was maintained at 34-39°C throughout these measurements. Cultures were returned to the incubator, as appropriate, between observations. Both the stage micromanipulator (Axiovert 135 inverted microscope; Carl Zeiss), and the Kontron image display, assisted in the accurate localization of the cells at each time point.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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). Here, we have applied a 96-h myosin IIB oligonucleotide treatment window to cells that had already been allowed to extend processes for various times up to 120 h. An underlying retractive process was uncovered upon application of the myosin IIB antisense treatment window (Figure 1), whereas sense, scrambled, or untreated controls had no effect. Process retraction eventually reached a minimum length of around 20 µm, whereupon further retraction was halted. The effects of the antisense regime were reversible, evident when the medium was replaced with one lacking oligonucleotides after a 96-h antisense treatment subsequent to unhindered outgrowth for 48 h (Figure 1a). When this underlying retractive process was examined with greater temporal precision by following individual cells and taking measurements at 6-8 hourly intervals after the application of myosin IIB oligonucleotides at either 72 h (Figure 1b) or 96 h (Figure 1c) after initiation of outgrowth, the actual rate of retraction could be measured. It was found to be relatively rapid, 50% of complete retraction ensuing within 
8 h (i.e., 1-2 µm/h) (Figure 1, b and c).
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Antisense Oligonucleotides Targeting Both Myosin IIA and Myosin IIB Prevent Neurite Outgrowth and Retraction in Cultured Neuro-2A Cells
After an initial delay, required for the antisense treatment to take full effect, simultaneous application of both myosin IIA and myosin IIB antisense oligonucleotides to Neuro-2A cells at the time of plating was found not only to suppress neurite extension as expected but also to prevent retraction (Figure 2a). The recovery phase subsequent to oligonucleotide removal was significantly delayed relative to that of cells treated with myosin IIB antisense alone (Wylie et al., 1998). If myosin IIA antisense oligonucleotides were provided continuously from the time of plating and myosin IIB antisense oligonucleotides were added only 96 h later, neurite retraction in the remaining adherent cells (Wylie and Chantler, 2001) was virtually abolished (Figure 2b). This contrasts with the behavior of cells given myosin IIB antisense treatment alone (Figure 1).
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Note that there is an alteration in phenotype of the double antisense-treated cells compared with their untreated controls (Figure 2, c and d). Whereas both neurite outgrowth and retraction were attenuated in these cells, lamellipodial outgrowth continued, leading to aberrant, expansive, and veil-like processes. This underlines the fact that, to a first approximation, lamellipodial dynamics and neurite outgrowth operate by different mechanisms.
Effect of Antisense Oligonucleotides Targeting Myosin IIA on the Neuro-2A Cytoskeleton
Because our previous results had shown that suppression of myosin IIA for 96 h could be disruptive to the actin cytoskeleton (Wylie and Chantler, 2001), we were concerned that the effects of myosin IIA antisense on retraction might be secondary to such induced disarray. We therefore decided to generate retractive responses to agonists applied after a relatively brief incubation period with myosin IIA oligonucleotides. To observe whether a 48-h myosin IIA antisense pretreatment was disruptive to the cell cytoskeleton, we compared the actin cytoskeleton of sense-treated and antisense-treated cells after 48 or 96 h by using rhodaminephalloidin staining (Figure 3). No differences could be discerned between the staining patterns of sense-treated cells at either 24 h (our unpublished data) or 48 (Figure 3) h with those of antisense-treated cells at similar times. Disruption of the actin cytoskeleton and attenuation of myosin IIA and paxillin immunofluorescence were observed at 96 h, consistent with previous observations (Wylie and Chantler, 2001).
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LPA-induced Retraction of Neuritic Processes in Cultured Neuro-2A Cells Is Inhibited by Antisense Oligonucleotides Targeting Myosin IIA but Not Those Targeting Myosin IIB
To examine whether LPA was able to induce neurite retraction in Neuro-2A cells, as has been observed in other cells of neuronal origin (Tigyi and Miledi, 1992; Jalink et al., 1993), Neuro-2A cells were grown in culture for 72 h before addition of LPA (1 µM). This pulse of LPA induced rapid process retraction that was virtually complete within 30 min (Figure 4a). This effect could be blocked entirely by prior addition of the Rho-kinase inhibitor Y27632 (25 µM), 30 min before LPA application (Figure 4b).
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LPA-induced retraction was also blocked through preincubation, for 48 h, with antisense oligonucleotides targeting myosin IIA (Figure 4d). Pretreatment with control sense (Figure 4c) or scrambled (our unpublished data) oligonucleotides targeting myosin IIA had no effect on LPA-induced retraction. In contrast, pretreatment with antisense oligonucleotides directed against myosin IIB sequence for a period of 18 h before agonist addition (sufficient time to ensure that at least 60% of the initial neurite length remained in place for LPA challenge) had very little inhibitory effect on LPA-induced retraction (Figure 4f). Here, process withdrawal was 80% of that seen in the presence of myosin IIB sense (Figure 4e) or scrambled (our unpublished data) oligonucleotides.
Antisense Oligonucleotides Targeting Myosin IIA but Not Those Targeting Myosin IIB Inhibit Thrombin-induced Retraction of Neuritic Processes in Cultured Neuro-2A Cells
We also determined that thrombin could induce neurite retraction in Neuro-2A cells, as has been observed in other cells of neuronal origin (Jalink and Moolenaar, 1992; Suidan et al., 1992). Thrombin (pulse optimized at a concentration of 5.0 NIH units/ml, as determined from a dose-response curve) caused immediate retraction of preformed neurites (Figure 5a). This could be blocked completely by Y27632 (25 µM) (Figure 5b).
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Whereas pretreatment with sense (Figure 5c) or scrambled (our unpublished data) oligonucleotides derived from myosin IIA sequences had no effect, a 48-h pretreatment with antisense oligonucleotides targeting myosin IIA again suppressed thrombin-induced retraction (Figure 5d) similar to results obtained with LPA. In contrast, antisense oligonucleotides targeting myosin IIB sequence had only a minimal effect on thrombin-induced retraction (Figure 5f), process withdrawal reaching 80% of that observed in the presence of sense (Figure 5e) or scrambled (our unpublished data) control oligonucleotide levels.
Y27632 Does Not Inhibit Neurite Outgrowth
Having used Y27632 to inhibit neurite retraction (Figures 4 and 5), we also examined whether this Rho kinase inhibitor had any effect on neurite outgrowth. Application of Y27632 to Neuro-2A cells at various times after plating demonstrated no inhibition of neurite outgrowth (Figure 6a). Indeed, the rate of outgrowth was somewhat enhanced. In a separate series of experiments, Y27632 was applied continuously from the time of plating then antisense oligonucleotides directed against myosin IIB were added at later times (Figure 6b). Under these conditions, myosin IIB antisense oligonucleotides remained effective at halting outgrowth, irrespective of the time of application. These results further suggest that Y27632 and myosin IIB antisense oligonucleotides interfere with separate pathways and thereby imply that retraction and outgrowth, respectively, involve distinctive molecular motors regulated by separate pathways.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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; Letourneau et al., 1991; Gordon-Weeks, 2000; Luo, 2002). Net growth evolves from a balance of forces, the mechanism involving alternation between periods of outgrowth and retraction, attachment and detachment. Each of these operational modes is presumably determined by the actions of one or more molecular motors, the activities of which must be coordinated to generate a resultant displacement in the required direction. We have shown previously that the molecular motors, myosin IIB and myosin IIA, undertake different roles in this process: myosin IIB drives outgrowth (Wylie et al., 1998), whereas myosin IIA maintains tensile adhesion (Wylie and Chantler, 2001). Here, we show that myosin IIA also drives neurite retraction.
By introducing isoform-specific myosin IIB oligonucleotides at different times after initiation of neurite outgrowth rather than continuously from the time of plating, as described previously (Wylie et al., 1998), we were able to observe an underlying process of continual neurite retraction (Figure 1). This retraction is, in turn, inhibited by antisense nucleotides directed against myosin IIA, which is thus the agent of the retraction process. Most striking, our observations are consistent with a constitutive retraction process that requires myosin IIA but is independent of myosin IIB. It is of interest to note that retraction is never complete, but ceases at neurite lengths 10-20 µm from the cell body (Figure 1). This limit may represent a differentiative boundary below which committed neurites can no longer shorten, an interpretation consistent with different roles for upstream regulators during neurite initiation compared with neurite elongation (Sebok et al., 1999).
Initial outgrowth up to 20 µm occurs in the presence of myosin IIB antisense oligonucleotides (Wylie et al., 1998), even when antisense oligonucleotides against both myosin IIA and myosin IIB are present from the time of plating (Figure 2a). Once this minimal length is attained, however, both antisense actions become effective so that outgrowth and retraction are equally constrained. Similarly, continued outgrowth of adherent cells in the chronic presence of antisense oligonucleotides targeting myosin IIA is curtailed upon addition of myosin IIB antisense oligonucleotides (Figure 2b), but retraction does not supervene. These results (Figure 2, a and b), together with data from the myosin IIB antisense window experiments (Figure 1), suggest that myosin IIA is important for neurite retraction. Despite the complete cessation of neurite outgrowth and retraction that accompanies treatment with both antisense reagents, lamellipodial expansion continued, yielding aberrant club-like structures (Figure 2, c and d). Thus, the dynamics of neurite extension (Wylie et al., 1998; Bridgman et al., 2001; Wylie and Chantler, 2001) and lamellipodial protrusion (Rochlin et al., 1995; Wang et al., 1996; Diefenbach et al., 2002) are, by implication, controlled by separate mechanisms.
To exclude the possibility that myosin IIA antisense effects arise from disruption of the actin cytoskeleton, we used LPA or thrombin to induce rapid neurite retraction in Neuro-2A cells that had been incubated with oligonucleotides for too brief a time to cause cytoskeletal disruption (Figures 3, 4, 5). Observation of the actin cytoskeleton by confocal microscopy did not identify any disturbance of the cytoarchitecture as a consequence of the relatively brief (48-h) incubation with myosin IIA antisense oligonucleotides (Figure 3). LPA and thrombin bind to different cognate heterotrimeric Ras family G-protein-coupled receptors; in either case binding engenders Rho activation, growth cone collapse and neurite retraction (Jalink et al., 1994; Tigyi et al., 1996; Katoh et al., 1998b). LPA-induced retraction of neurites from N1E-115 cells has been shown to be inhibited by Y27632 (Hirose et al., 1998), itself a specific inhibitor of Rho-kinase (also known as p160ROCK or ROK
). Rho-kinase is also known to induce neurite retraction (Hirose et al., 1998; Katoh et al., 1998a) and its action involves myosin II activation (Amano et al., 1998). Rho kinase, activated by Rho, phosphorylates and thus inhibits myosin light chain phosphatase, leading to the persistence of myosin II-actin interaction. Y27632 inhibits the action of Rho-kinase; phosphatase activity is consequently preserved and the myosin regulatory light-chain is dephosphorylated, thereby terminating cross-bridge cycling.
Our data show that both LPA- and thrombin-induced Neuro-2A cell neurite retraction, which reaches completion within 30 min of addition of agonist, can be inhibited by prior exposure to antisense oligonuclotides directed against myosin IIA transcripts (Figures 4 and 5). Inhibition is essentially complete (Figures 4d and 5d), as also seen when Y27632 alone is added (Figures 4b and 5b). Most importantly, antisense-dependent inhibition is isoform-specific: oligonucleotides that suppress myosin IIB transcripts have a minimal, albeit finite, effect on neurite retraction irrespective of whether growth cone collapse is induced by LPA (Figure 4f) or by thrombin (Figure 5f).
Two possible interpretations arise to explain why myosin IIB antisense oligonucleotides have a small effect (Figures 4f and 5f), rather than none at all. Either myosin IIB could act, in part, as a retraction motor or, alternatively, the action of myosin IIA requires normal myosin IIB functionality. The observation that myosin IIA antisense oligonucleotides alone inhibit retraction completely (Figures 4d and 5d), similar to Y27632 (Figures 4b and 5b), argues against the former, because if true, one would not expect the effect of myosin IIA antisense alone to be maximal. Even though the LPA (Figure 4a) and thrombin (Figure 5a) stimuli for retraction used in these experiments are themselves maximal, when in combination with the action of antisense oligonucleotides targeting myosin IIB suboptimal retraction is observed (Figures 4f and 5f). Consequently, it is likely that optimal IIA action is conditional upon normal IIB activity during retraction. This conditional linkage is not reciprocal in its action and is so far revealed only in the context of retraction. For example, suboptimal neurite outgrowth (requiring the myosin IIB motor) is not observed when cells are subject to myosin IIA antisense (Wylie and Chantler, 2001) or Y27632 (Figure 6). If the two motors were linked in a reciprocal manner, then suboptimal outgrowth would be predicted. Y27632 has no inhibitory effect on neurite outgrowth (Figure 6a) and does not antagonize attenuation of outgrowth by antisense oligonucleotides targeting myosin IIB (Figure 6b). The small increase in the rate of neurite outgrowth that accompanies Y27632 addition (Figure 6, a and b) is likely due to a rearrangement of the actin cytoskeleton arising as a consequence of myosin IIA inhibition downstream of the Rho-kinase inhibitor. Thus, the vectorial balance of forces will be altered, facilitating an increased rate of outgrowth powered by myosin IIB.
These results again reveal the separate roles performed by the distinct yet closely homologous isoforms, myosins IIA and IIB (Wylie and Chantler, 2001). They show, for the first time, that myosin IIA but not myosin IIB drives growth cone collapse and neurite retraction. Our data, together with the work of others (Amano et al., 1998; Hirose et al., 1998; Katoh et al., 1998a), place the regulation of myosin IIA downstream of Rho and Rho-kinase activity and imply that a different pathway regulates the reciprocal functions of myosin IIB. It is likely that this latter control pathway is regulated upstream by Rac, the overexpression of which is known to induce neurite outgrowth (Albertinazzi et al., 1998) and which has been shown to regulate the phosphorylation state of the myosin heavy chain (van Leeuwen et al., 1999). We infer that the target for heavy chain phosphorylation is myosin IIB, the isoform that has previously been shown (Wylie et al., 1998; Bridgman et al., 2001) to be required for neurite outgrowth.
The above-mentioned results allow us to construct an overall scheme (Figure 7) for the separate regulation of myosin IIA and myosin IIB functions, one which also incorporates results from several other laboratories (Ridley, 1996; Sanders et al., 1999; van Leeuwen et al., 1999; Bishop and Hall, 2000). Myosin IIB activity, responding to external agonists, such as bradykinin or nerve growth factor, is necessary for neurite outgrowth (Wylie et al., 1998; Bridgman et al., 2001) and cell spreading (Diefenbach et al., 2002). Myosin IIA activity, responding to a variety of external agonists, including LPA and thrombin, is required for neurite retraction (this study) and focal contact formation (Wei and Adelstein, 2000; Wylie and Chantler, 2001) and is likely to be the isoform involved in stress fiber formation (Chrzanowska-Wodnicka and Burridge, 1996). Communications between pathways have been found that hint at reciprocal upstream control of myosin II isoform activity. These include mutual antagonistic effects of Rho relative to both Rac and Cdc42 (Hirose et al., 1998; Rottner et al., 1999; Sander et al., 1999; Sanders et al., 1999; Bishop and Hall, 2000; Yuan et al., 2003), reciprocity of activation of neurite outgrowth and inhibition of neurite retraction or cell rounding by cAMP (Gunning et al., 1981; Tigyi et al., 1996a; Hirose et al., 1998), or PAK (Sanders et al., 1999; van Leeuwen et al., 1999). The two pathways are likely to respond differently to extracellular cues during axonal pathfinding.
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The integrated mechanism by which both myosin IIA and myosin IIB contribute to neurite elongation will be discussed elsewhere (Chantler and Wylie, 2003). However, it is worth noting that the differential localization of myosin IIB and myosin IIA (Rochlin et al., 1995; Kolega, 1998), together with the distinct spatial organization of actin cytoarchitecture with the growth cone (Bridgman and Dailey, 1989; Lewis and Bridgman, 1992), are critical to their actions. The separate but linked functions of myosin IIA and myosin IIB, together with their distinct localization, lead one to speculate that an additional tier of their control may reside in compartmentalization of key regulatory signals. For example, myosin IIA is required for both neurite retraction and for focal contact formation; whereas retraction must involve extensive breakdown of existing contacts, outgrowth, while dependent on myosin IIB, can only occur with participation of adhesive forces. It is possible, therefore, that activation of myosin IIA is spatially restricted, focal contact formation being stimulated by local signaling through the matrix binding sites.
Although many details remain to be clarified, we conclude from our work that two highly conserved conventional myosin motors exhibit separate and complementary functions, acting in response to distinct regulatory pathways: myosin IIB powering neurite outgrowth, and myosin IIA driving neurite retraction and facilitating adhesion. Their actions combine to generate the vectorial forces required for neurite extension.
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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* Corresponding author. E-mail address: pchant{at}rvc.ac.uk.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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